Association between zinc deficiency and aorta stiffness in non-diabetic hemodialysis patients

Objectives Zinc deficiency (Zn < 60 μg/dL) is known to play an important role for vascular calcification. However, little data is available regarding the association between zinc deficiency and aorta stiffness in dialysis patients. Thus, we studied the relationship between zinc deficiency and aorta stiffness in non-diabetic hemodialysis (HD) patients. Methods Of 150 patients receiving maintenance HD at our hospital, we included 79 non-diabetic HD patients (age: 70±11 years, 49 men) after excluding 71 diabetic HD patients. Zinc deficiency was defined as Zn <60 μg/dL during pre-HD blood sampling. The association between zinc deficiency and aorta stiffness was analyzed. Aorta stiffness was evaluated as brachial-ankle pulse wave velocity (baPWV). Other surrogate markers for cardiovascular complications were also measured. Results The zinc deficiency group (ZD group) included 45 patients (57.0%). Compared to the zinc non-deficiency group (ZND group), patients with ZD group were significantly older, higher levels of CRP and hypoalbuminemia. Moreover, they had significantly higher levels of baPWV, and lower levels of ankle-brachial pressure index (ABI) (p<0.05). After adjusting for hypoalbuminemia, and CRP, multivariate analysis showed that age and zinc level were independent predictors of baPWV. Conclusion The study suggested that zinc deficiency may be an independent risk factor for aorta stiffness, even after adjusting for malnutrition and inflammation.


Introduction
Dialysis patients are characterized by a high risk of developing cardiovascular disease (CVD) events such as ischemic heart disease, cerebrovascular accident, and heart failure, and a low survival rate after CVD [1]. Vascular calcification, which is the cause of CVD, progresses even before the introduction of hemodialysis. There are two types of calcification: atherosclerotic calcification lesions, which are mainly in the intima, and Mönkeberg-type calcification lesions, which are mainly in the tunica media. The latter is predominant in dialysis patients [2], the tunica media calcification has been reported to be related to arterial stiffness.
The pulse wave velocity (PWV) of the aorta is a representative index of arterial wall stiffness and a predictor of the risk of CVD death and total mortality in dialysis patients [3]. Among them, brachial-ankle peak wave velocity (baPWV) is one of the most important surrogate markers for arterial wall stiffness, as it can be easily measured between the brachial joint and the ankle joint. High levels of baPWV have been known to be a prognostic factor of all-cause mortality and cardiovascular mortality. for hemodialysis patients [4].
Zinc is one of the nine essential trace elements in humans, and is involved in the function of over 300 types of enzymes, cytokines, hormones, etc. It regulates the activation and signal transduction mechanisms involved in intracellular metabolism and cell response in nutrient metabolism and in the central nervous, immune, endocrine, digestive, and circulatory systems [5,6].
Recent reports indicate that zinc plays an important role in suppressing the calcification of vascular smooth muscle [11,12]. Vascular calcification is serious problem, leading to poor prognosis in terms of cardiovascular events, particularly in patients with chronic kidney disease [13,14]. However, to our knowledge, there are no reports on the association between zinc and aorta stiffness in dialysis patients. Therefore, we investigated the association between serum zinc levels and aorta stiffness in HD patients excluding those with diabetes because of ever well-known strong risk factor for aorta stiffness.

Subjects (Fig 1)
All patients gave informed consent, and local ethical committee (the Tokushukai Group Institutional Review Board) approved the study (ethical No.TGE 01845-024) and was performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained through an opt-out procedure; patients were provided with information explaining the proposed research project (purpose and required individual data) by means of an information sheet or a webpage on the hospital's website and were given the opportunity to opt you. Demographic, clinical and laboratory data were retrieved from medical records. All data were fully anonymized.
Of all 150 maintenance HD patients who were outpatients at the Shonan Kamakura General Hospital Dialysis Center, 71 diabetic patients were excluded, and 79 patients were included in the study (Fig 1).

Zinc measurements and analysis items
Zinc measurements were conducted by promptly centrifuging the collected blood simultaneously with regular blood sampling (before dialysis at the beginning of the week, which was conducted after a 2-day interval), and the serum zinc levels were measured in our facility using the atomic absorption spectrophotometric method.
We analyzed relationships between serum zinc levels and the following: patient background (sex, age, body mass index, dialysis history, presence of diabetes), dialysis method (HD/online hemodiafiltration), regular blood sampling test items (39 in total), echocardiography parameters (ejection fraction, left ventricular mass index, E/e', relative wall thickness, presence of valve calcification), and physiological tests (ankle-brachial index [ABI], toe-branchial index, baPWV, skin perfusion pressure [SPP]). The body mass index calculation used the estimated ideal weight (dry weight) at the time of regular blood sampling. Analyses were also conducted by dividing the group into those with Zn < 60 μg/dL, and those with Zn � 60 μg/dL, which is one of the diagnostic criteria for zinc deficiency. baPWV/ABI was measured on non-dialysis days with Form PWV/ABI (OMRON Colin, Tokyo, Japan). SPP, which is used for detecting microcirculatory impairment, was measured at the same time with laser-doppler type device (PAD 4000 1 , Kaneka Co.,Ltd, Tokyo, Japan).

Statistics
Continuous variables were compared using the Mann-Whitney U-test. The selection of explanatory variables for multiple regression analysis and binary logistic regression analysis were conducted using the stepwise variable increase/decrease method and variable increase method (likelihood ratio), respectively. The statistical analysis software used was JMP 10 (SAS Institute, JAPAN). The statistical significance level was set at p < 0.05.

Patient background
The 79 (49 men, 62%) patients that were analyzed in this study had a median age of 70 (IQR 62-79) years, and a median hemodialysis duration of 6.9 (IQR 2.3-15.6) years.
The levels of baPWV were significantly higher in the ZD group (1958±748 vs. 1727±441, p = 0.011) ( Table 2). As shown in Fig 3, there is a significant correlation between baPWV and

Multivariate analysis
To examine factors associated with baPWV, a multivariate analyses was conducted with baPWV as the dependent variable and the results of blood test showing significant differences between the ZD group and ZND group as explanatory variables (Table 3). We found that zinc were independent predictors of baPWV (β = -9.85, t value = -2.00, p = 0.047) as well as older age (Table 3).

Discussion
Our study clearly showed that zinc level was an independent predictive factor of baPWV, which is an indicator of aorta stiffness in HD patients. Risk factors for atherosclerosis and CVD in CKD patients include classic risk factors such as diabetes, old age, male sex, hypertension, and smoking, as well as the non-classic risk factors such as anemia, oxidative stress, inflammation, and malnutrition [15]. Our present results indicate that hypozincemia is associated with aorta stiffness in HD patients.
The onset and progression of atherosclerosis and CVD begin from early stages of CKD. Atherosclerotic changes in the coronary arteries, particularly coronary artery calcification, are noted before the introduction of dialysis; when the estimated glomerular filtration rate (eGFR) drops below 45 mL/min/1.73 m 2 , both the prevalence and extent of calcification significantly increase [14]. Coronary artery stenosis has been frequently observed during the introduction of dialysis, affecting 53% of asymptomatic CKD patients and 83% of diabetic CKD patients [16]. Several studies have reported that zinc plays an important role in suppressing the calcification of vascular smooth muscle cells (VSMCs). Voelkl et al. reported that zinc sulfate 1)  suppressed phosphate-induced calcification, 2) decreased the expression of mRNA (which is a bone formation marker that includes the expression of zinc-finger protein TNF-α-induced protein 3 (TNFAIP3)), and 3) inhibited NF-kB activation and bone/cartilage reprogramming, and consequently suppress VSMC calcification by phosphate [11]. Therefore, serum zinc levels should be carefully maintained. Indeed, it is reported that increased dietary intake of zinc decrease 8% reduction of aorta calcification [17].
In the present study, serum zinc deficiency was associated with aorta stiffness independent from serum albumin levels. The fact was similar to as the report that zinc deficiency was associated with 2-year mortality in HD patients [18]. Therefore, we need to consider the other factor for this association. Inflammation might be an important factor because malnutritioninflammation-atherosclerosis (MIA syndrome) is well-known as a central axis [19]. In this regard, however, CRP values were not chosen as independent factor.
Causes of zinc deficiency in dialysis patients include inadequate zinc intake, impaired absorption, and loss of zinc to the dialysate solution [10]. In this study, albumin, prealbumin, creatinine, and GNRI were significantly lower in the zinc-deficient group, but the percentage of on-line HDF was lower in the zinc-deficient group (29.6% vs. 48.6%). Although a nutritional survey was not available, none of the subjects in this study used zinc products, so the main cause of zinc deficiency could be inadequate zinc intake.
Peripheral arterial disease (PAD) is one of serious complications among HD patients [20]. ABI is used as a screening test for the diagnosis of PAD. Also, vascular calcification plays an important role for the pathophysiology of PAD. The present study showed that ABI in ZD group was significantly lower than that of ZND group. Bérard, et al show that in dialysis patients zinc deficiency had 3.4-fold increase in development of PAD [21].
There are several limitations to this study. First, this was a cross-sectional observational study. Second, this study was conducted in a single facility and had a small sample size. Third, diabetic HD patients were excluded because of the greater risk factor of atherosclerosis, other risk factors for atherosclerosis in CKD patients were not sufficiently excluded. This study excluded diabetes, which is a major risk factor for aorta stiffness; however, classic risk factors such as hypertension and nutritional evaluation/lack of exercise were either not excluded or not incorporated into this study.
In conclusion, this single-center cross-sectional study showed that zinc levels in HD patients was an independent predictor of baPWV, which is an indicator of aorta stiffness. Further study is needed to clarify whether hypozincemia worsens the prognosis of HD patients.